Optimizing bacteriophage engineering through an accelerated evolution platform

Abstract

The emergence of antibiotic resistance has raised serious concerns within scientific and medical communities, and has underlined the importance of developing new antimicrobial agents to combat such infections. Bacteriophages, naturally occurring bacterial viruses, have long been characterized as promising antibiotic alternatives. Although bacteriophages hold great promise as medical tools, clinical applications have been limited by certain characteristics of phage biology, with structural fragility under the high temperatures and acidic environments of therapeutic applications significantly limiting therapeutic effectiveness. This study presents and evaluates the efficacy of a new accelerated evolution platform, chemically accelerated viral evolution (CAVE), which provides an effective and robust method for the rapid enhancement of desired bacteriophage characteristics. Here, our initial use of this methodology demonstrates its ability to confer significant improvements in phage thermal stability. Analysis of the mutation patterns that arise through CAVE iterations elucidates the manner in which specific genetic modifications bring forth desired changes in functionality, thereby providing a roadmap for bacteriophage engineering.

Figure 1

Schematic of the CAVE pipeline for bacteriophage directed evolution, with iterative cycles of mutagenesis and thermal selection. In this procedure, bases within the bacteriophage genomes are randomly affected by treatment with a chemical mutagen. Upon replication within a host, a mismatch mutation is introduced. The mutant library is then subjected to high-temperature incubation to select for thermally stable variants; surviving phages are amplified and carried over to subsequent iterations.

Figure 2

Improvement of thermal stability through directed evolution. (a) Multiple rounds of this procedure leads to increased resistance to thermal degradation. Incubation of T3 phages at 60 ${}^{\circ }$C for 1 h resulted in active phage survival of 6.6%, 36.0%, 59.1%, and 69.9% after 0, 10, 20, and 30 rounds of directed evolution, respectively. Relative concentrations were calculated using plaque-counting assays and linear regressions over four 40:140 $\mathrm{\mu }$L serial dilutions (n = 4); error bars representing ± SD. (b) The effect of temperature on degradation rate. The percent survival of wild type and mutant bacteriophages over the course of a 70 min incubation at 60, 62, and 64 ${}^{\circ }$C, quantified using a linear regression over serial dilutions (n = 3). Curves were fit to Eq. (1). Error bars represent the difference between theoretical curve values and measured titer for a given time point. (c) Mutant phages display improved tolerance to acidic conditions. Percent survival after 30 min incubation under acidic conditions, relative to neutral control conditions. (d) Half life values calculated for wild type and mutant phages at 25, 60, 62, and 64 ${}^{\circ }$C; error bars are ± SD. Relative concentrations were calculated using plaque-counting assays and linear regressions, with error bars representing ± SD. Theoretical curves were fit to experimental data using a fractional-order kinetic model and the Arrhenius law.

Figure 3

Genomic changes observed in the T3 bacteriophage genome over the course of 30 applications of the CAVE protocol. (a) Mutation frequencies observed in the T3 mutant gene pools after 10, 20, and 30 rounds of directed evolution. Mutation-frequency bars are color-coded corresponding to their functional genome location where they occur: blue are non-coding regions, magenta are structural genes, and purple are regulatory genes. (b) Fraction of net mutation frequencies observed within structural genes, regulatory genes, or non-coding regions, combined from final evolved generations of all T3 mutants from first and second CAVE series. (c) Comparison of mutation frequency indices at round 20 of CAVE application for two different series of T3 mutagenesis. (d) Protocol for recapitulation of mutations at specific sites using phage-rebooting. (e) Results from recapitulation of mutations at several structural genes verify that such mutations confer improved stability.

Figure 4

Application of CAVE protocol to other bacteriophage species. (a) Comparison of wild type vs. mutant T7 bacteriophage (after 15 rounds of directed evolution), percent survival following 1-h incubation at 25 ${}^{\circ }$C and 60 ${}^{\circ }$C. (b) Mutation frequencies observed in the mutant gene pools of the T7 bacteriophage after 5, 10, and 15 rounds of directed evolution. (c) Comparison of mutation frequency indices at round 20 of CAVE application between bacteriophages T3 and T7. (d) Crystal structures of the assembled T7 tail complex (PDB ID = 6R21), with high mutagenesis proteins: head-to-tail joining protein, tail-tubular protein A, tail-tubular protein B (mutation sites colored in magenta). (e) Salmonella phages NBSal001 and NBSal002: comparison of wild type vs. mutant (after 15 rounds of directed evolution), percent survival following 1-h incubation at 25 ${}^{\circ }$C and 60 ${}^{\circ }$C.